1. Field of the Invention
The present invention relates generally to capillary columns having interior surface coatings and electrophoretic separation methods for their use. More particularly, the present invention involves capillary columns having a neutral crosslinked hydrophilic coating on their interior wall surfaces. The neutral crosslinked hydrophilic coating reduces analyte interaction with the interior surface of the capillary column and simultaneously protects the interior surface of the capillary column during the electrophoretic separation of acidic and basic compounds.
2. Description of Relevant Art
Electrophoretic separation techniques have been utilized for years to separate molecules according to differences in the effective charge of the molecules, and/or according to differences in the molecular size of the molecules. Up until recently electrophoretic separations were conducted in gel slabs or open gel beds which were typically fabricated of polyacrylamide gel material. More recently capillary electrophoresis techniques combined with photometric detection methods have allowed the automation and rapid quantitative analysis of molecules. High resolution separations of molecules having different effective charges have been achieved by applying electrophoretic principles to buffer filled or gel filled narrow capillary tubes.
Typically, capillary columns used in capillary electrophoresis are fabricated of lengths of silica tubing having diameters on the order of 25 .mu.m to 200 .mu.m and lengths from about 10 to 200 cm. The buffer and gel separation mediums are pumped directly into the column interiors and electrophoretic techniques are used to separate numerous types of molecules including peptides, proteins, and oligonucleotides, nucleic acids and other charged molecular species. The field of electrophoretic separation technology is continually expanding with respect to the types and sizes of molecules which can be separated and detected using capillary electrophoresis procedures.
The advantages associated with capillary electrophoresis are numerous. Quantitative information can be achieved with very small sample sizes, and the amount of gel or buffer consumed is minuscule. Furthermore, the time required for the separations is sharply reduced, and the technique lends itself to automation and electronic data storage and data manipulation. Significantly, capillary electrophoresis is associated with certain phenomenon which are not present in traditional slab gel electrophoresis. One of these is the now familiar electroosmotic flow phenomenon characterized by bulk flow of buffer solutions toward one of the electrodes.
Electroosmotic flow is generated by the ionization of silanol functionalities on the surface of silica capillary tubing. The ionization results in a layer of protons in the electrophoretic buffer solution at the surface of the silica tubing. In the presence of an electric field the layer of protons resembles a positively charged column of fluid which migrates toward the cathode, causing a general bulk movement of the buffer medium. Advantageously, electroosmotic flow can be utilized in many applications to improve electrophoretic separations. For example, when the electrophoretic migration of the molecules being separated is in the opposite direction to that of electroosmotic flow, the net effect is an increase in column performance and improved separations.
For many electrophoretic applications electroosmotic flow is undesirable and eliminating or substantially reducing the bulk flow is preferred. Generally, when electroosmotic flow is reduced to a minimum, electrophoretic sample components move only by electrophoretic migration, which improves analysis reproducibility and mass recovery of sample components.
One method to minimize or to control electroosmotic flow, is to utilize capillary columns fabricated of silica capillary tubing coated on the inside with a polymeric material. The polymeric coating eliminates or substantially reduces the degree of ionization of the surface silanol groups which causes at least a substantial reduction in the bulk flow within the electrophoresis column. In order to avoid unwanted hydrophobic-hydrophobic interactions between sample components and the coating, the polymeric coatings are traditionally hydrophilic. One problem associated with the nature of prior art hydrophilic polymeric coatings is their low physical integrity, their tendency to dislodge from the surface of the capillary, and the resulting short useful life. Another problem with prior art hydrophilic polymeric coatings relates to their photo stability. More particularly, these coatings tend to absorb uv radiation at wavelengths below 260 nm and are subject to photolysis which causes their eventual loss of functionality. This is particularly undesirable in analytical electrophoresis systems using uv detectors.
Covalently binding hydrophilic polymers to the surface of the capillary tubing, helps reduce the coating's tendency to dislodge, however the physical integrity and photo stability of the coatings remains a problem.
Additionally, even when covalently bound to the surface of silica capillaries through an Si-O-Si, prior art hydrophilic coatings promote aqueous interaction with Si-O-Si functionalities. This can result in the unwanted hydrolysis and the Si-O-Si bond and a resulting shorted use life time.
As mentioned above, another problem associated with capillary electrophoresis techniques is the tendency for sample components to adhere to the wall of the capillary tubing, and in particular silica tubing. This is especially true in the case of small charged molecules which are easily attracted to reactive silica functionalities. When small peptides and amines are present in electrophoretic separation mediums, they interact both electrostatically and hydrophobically with the capillary wall. The result is a decrease in separation efficiency and undesirable band broadening which gives erroneous separation data.
Like electroosmotic flow, providing electrophoresis capillaries which are capable of minimizing the degree of sample component and wall interaction have not been totally successful. Previous attempts include using a dynamic double layer coating. These bilayer coatings are not stable and require additives in the running buffers used during the electrophoretic process. Hydrophobic coatings are effective in protecting the Si-O-Si surfaces of silica columns, but cause unwanted hydrophobic-hydrophobic interactions between the capillary wall and analytes. As noted above, hydrophilic coatings are not effective in protecting the Si-O-Si surface.
Accordingly, it is an objective of the present invention to provide novel coated capillary columns useful for electrophoresis separations and which minimize electroosmotic flow.
It is additionally an objective of the present invention to provide capillary columns which reduce or eliminate interactions between sample components and the interior capillary wall.
It is further an objective of the present invention to provide capillaries having physically stable coatings.
It is additionally an objective of the present invention to provide capillaries useful for the electrophoretic separation of a variety of charged molecules.
It is another objective of the present invention to provide coated capillary columns having improved useful life when used in connection with uv detectors.